Supplemental material



Supplemental data for:

Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step

Elisabeth J. Chapman1, Alexey I. Prokhnevsky1, Kodetham Gopinath1,2, Valerian V. Dolja1,3 and James C. Carrington1,3,4

1Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA

2Present address: Biochemistry & Biophysics, Texas A&M University, College Station, TX 77843, USA

3Center for Gene Research and Biotechnology, Oregon State University, Corvallis, OR 97331, USA

4Corresponding author. e-mail: Carringtoncarrington@orstcgrb.oregonstate.edu

Results

To confirm that the HA-tagged viral proteins used in this study were functional RNA silencing suppressors, we analyzed each construct in Agrobacterium-mediated transient assays in Nicotiana benthamiana leaves (Johansen and Carrington 2001). This assay involves co-expression of constructs encoding functional GFP, a potent inducer of GFP RNA silencing [a hairpin-generating construct (dsGFP)], and a silencing suppressor. In each assay, apposing half-leaves were infiltrated with mixtures that either contained (experimental sample) or lacked (control sample) the suppressor construct. Silencing was analyzed by inspection of visual GFP fluorescence, detection of GFP protein and mRNA, and detection of GFP-specific siRNAs. Coexpression of GFP and dsGFP constructs without a silencing suppressor resulted in no or low levels of GFP fluorescence (Fig. S1A) and low or non-detectable levels of GFP mRNA and protein (Fig. S1B and S1C, lanes 5 and 6). Tissue expressing only GFP and dsGFP constructs also accumulated GFP-specific siRNAs of 21-24 nucleotides in length (Fig. S1D, lanes 5 and 6). Each of these features is characteristic of efficient RNA silencing of the GFP construct in the infiltration zone.

In contrast to the controls, RNA silencing was suppressed effectively in leaves that co-expressed each of the modified silencing suppressors P1/HC-Pro, p21, p19 and CP (Supplemental Fig. S1A-C). Fluorescence was detected, and GFP protein and GFP mRNA accumulated to relatively high levels. P1/HC-Pro, p21 and p19 each failed to inhibit siRNA formation using dsGFP as the silencing inducer (Supplemental Fig. S1D), indicating that these suppressors likely function to inhibit one or more steps after siRNA formation. Suppression of siRNA accumulation by p19 and P1/HC-Pro was previously reported by several groups using sense transgenes as a silencing locus (Llave et al. 2000; Mallory et al. 2001; Hamilton et al. 2002; Mallory et al. 2002; Silhavy et al. 2002; Himber et al. 2003). In these cases, efficient silencing of the inducer locus may require RNA-dependent RNA polymerase-mediated production of a double-stranded RNA intermediate and subsequent amplification steps (Dalmay et al. 2000; Llave et al. 2000; Vaistij et al. 2002; Himber et al. 2003). Loss of siRNA accumulation in these cases may be an indirect effect of suppression of amplification (Llave et al. 2000; Himber et al. 2003).

The TCV CP suppressor inhibited siRNA formation, which is consistent with previous experiments using non-tagged versions in other assays (Qu et al. 2003). The CP suppressor likely interferes with the dsRNA processing step catalyzed by a DICER-LIKE activity. CMV 2b was less effective than the other suppressors in this assay. GFP fluorescence was detected, but protein and mRNA accumulated only to low levels (Supplemental Fig. S1A-C). Non-tagged forms of CMV 2b protein were shown previously to have incomplete suppressor activity in infiltrated leaves, and to inhibit intercellular spread of a mobile silencing signal (Guo and Ding 2002).

Materials and Methods

Agrobacterium-infiltration assays

Transient silencing assays in Nicotiana benthamiana were done as described (Johansen and Carrington 2001). Cultures of A. tumefaciens GV2260 were injected at the following concentrations: 35S:GFP, OD600 = 0.5; 35S:dsGFP-FAD2, OD600 = 0.1; 35S:P1/HC-Pro or other suppressor constructs, OD600 = 0.5. In assays lacking one or more of these components, strains containing 35S:GUS or 35S:vector were used to normalize each injection to a constant OD600 = 1.0. Infiltrated tissues were harvested 48 hours post-injection for photography and protein and RNA analyses. GFP fluorescence was visualized using long-wavelength ultraviolet light.

Protein and RNA blot analysis

Protein extracts were prepared from infiltrated N. benthamiana tissues and normalized for SDS-PAGE using the Bradford assay (Bio-Rad). Immunoblot analysis of total protein samples (10 (g) was done using anti-HA-peroxidase conjugate (Roche). Total RNA was extracted from independent pools of infiltrated tissue with Trizol reagent (Johansen and Carrington 2001). Low-molecular weight RNA was isolated with RNA/DNA Midi Kits (Qiagen). Blot hybridization of normalized total or low-molecular weight RNA (5 µg) was done as previously described (Llave et al. 2002) and hybridization intensities were quantified using a Phosphorimager (Molecular Dynamics). Radiolabeled probes for mRNAs were synthesized by random-primed labeling of cloned smGFP sequences (Johansen and Carrington 2001) with [32P]dATP (Feinberg and Vogelstein 1983).

Phenotypic analysis of transgenic Arabidopsis

Phenotypic effects of suppressor expression were quantified at defined growth stages: vegetative parameters, stages 5.00 – 5.10; reproductive defects, stages 6.10 – 6.90; fertility, stages 8.00 – 9.70 (Boyes et al. 2001). Mean area of third and fourth rosette leaves was determined using a Licor Model 3100 area meter. Sepals and other floral organs were measured with a Mitutoyo Model CD-6”CS digital calipers. All data were subjected to statistical analyses using the Student’s t-test.

References

Boyes, D.C., Zayed, A.M., Ascenzi, R., McCaskill, A.J., Hoffman, N.E., Davis, K.R., and Göorlach, J. 2001. Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants. Plant Cell 13: 1499-1510.

Dalmay, T., Hamilton, A., Rudd, S., Angell, S., and Baulcombe, D.C. 2000. An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus. Cell 101: 543-553.

Feinberg, A.P. and Vogelstein, B. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 257: 8569-8572.

Guo, H.S. and Ding, S.W. 2002. A viral protein inhibits the long range signaling activity of the gene silencing signal. EMBO J. 21: 398-407.

Hamilton, A., Voinnet, O., Chappell, L., and Baulcombe, D. 2002. Two classes of short interfering RNA in RNA silencing. EMBO J. 21: 4671-4679.

Himber, C., Dunoyer, P., Moissard, G., Ritzenthaler, C., and Voinnet, O. 2003. Transitivity-dependent and -independent cell-to-cell movement of RNA silencing. EMBO J. 22: 4523-4533.

Johansen, L.K. and Carrington, J.C. 2001. Silencing on the spot: induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system. Plant Physiol. 126: 930-938.

Llave, C., Kasschau, K.D., and Carrington, J.C. 2000. Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway. Proc. Natl. Acad. Sci. USA 97: 13401-13406.

Llave, C., Kasschau, K.D., Rector, M.A., and Carrington, J.C. 2002. Endogenous and silencing-associated small RNAs in plants. Plant Cell 14: 1605-1619.

Mallory, A.C., Ely, L., Smith, T.H., Marathe, R., Anandalakshmi, R., Fagard, M., Vaucheret, H., Pruss, G., Bowman, L., and Vance, V.B. 2001. HC-Pro Suppression of transgene silencing eliminates the small RNAs but not transgene methylation or the mobile signal. Plant Cell 13: 571-583.

Mallory, A.C., Reinhart, B.J., Bartel, D., Vance, V.B., and Bowman, L.H. 2002. A viral suppressor of RNA silencing differentially regulates the accumulation of short interfering RNAs and micro-RNAs in tobacco. Proc. Natl. Acad. Sci. USA 99: 15228-15233.

Qu, F., Ren, T., and Morris, T.J. 2003. The coat protein of Turnip crinkle virus suppresses posttranscriptional gene silencing at an early step. J. Virol. 77: 511-522.

Silhavy, D., Molnár, A., Lucioli, A., Szittya, G., Hornyik, C., Tavazza, M., and Burgyán, J. 2002. A viral protein suppresses RNA silencing and binds silencing-generated, 21- to 25-nucleotide double-stranded RNAs. EMBO J. 21: 3070-3080.

Vaistij, F.E., Jones, L., and Baulcombe, D.C. 2002. Spreading of RNA targeting and DNA methylation in RNA silencing requires transcription of the target gene and a putative RNA-dependent RNA polymerase. Plant Cell 14: 847-867.

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Figure Legends

Figure S1. Activity of epitope-tagged silencing suppressors. (A) Leaves of N. benthamiana infiltrated with cultures of Agrobacterium containing empty vector (V) or genes for expression of ß-glucuronidase (GUS), GFP, dsGFP, and HA-tagged silencing suppressors P1/HCPro (P1/HC), p21, p19, CP or 2b. (B) GFP mRNA accumulation in co-Agro-infiltrated tissues. Blot hybridization was done on total RNA samples prepared from leaf tissues that were infiltrated with the Agrobacterium strains shown in (A). Mobility position of the 1.5 kb RNA standard is shown. Average relative accumulation (RA) of GFP signal in duplicate samples, relative to that in lanes 5 and 6 (dsGFP-FAD2 + GFP) is shown. Average accumulation of GFP mRNA in a single duplicate samples from CP-infiltrated tissues is indicated, but blot data from only one sample are shown (lane 13). (C) GFP protein accumulation in co-Agro-infiltrated tissues. Immunoblot analysis was done on total protein samples prepared from infiltrated tissues described in (A). Mobility position of the 25-kDa protein standard is shown. (D) GFP-specific siRNA accumulation in co-Agro-infiltrated tissues. Blot hybridization was done using duplicate low molecular weight RNA samples (1 (g) prepared from the infiltrated tissues described in (A). Mobility positions of 24- and 21-nucleotide RNA standards are shown. Mean relative accumulation (RA) of signal in duplicate samples, relative to that in the control (lanes 5 and 6) is shown.

Figure S2. Quantitation of defects in transgenic Arabidopsis. (A) Quantitation of vegetative defects in vector-transformed and suppressor-expressing Arabidopsis plants. Mean values for all parameters are represented on each graph with standard deviations shown as error bars. p-values are represented as follows: ‡, p > 0.05; *, p ≤ 0.05. (B) Quantitation of reproductive defects in vector-transformed and suppressor-expressing Arabidopsis plants. Standard deviations and p-values are represented as in (A).

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